Fig 2: CMTR1-KD Impairs Dendritic Development Independent of Secretory Factors and RIG-I/MAVS-Activated Signaling(A) Schematic diagram of the co-culture experiment. Neurons on coverslips were co-cultured with siCTL or siCM1 neurons from DIV3 to DIV7. The neurons on dishes were harvested for western blotting.(B) The neurons on coverslips were fixed for MAP2 immunostaining and analyzed for the number and total length of dendrites.(C) PCR genotyping of E17.5 embryos from RIG-I heterozygous mating.(D) RIG-I-KO neurons infected with the designated lentivirus were immunostained for MAP2. MAVS-KO neurons were processed similarly.(E and F) The dendritic intersections, number, and length were determined. (B, E, and F) Data are mean ± SEM (n = 60 neurons from three cultures). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA. Scales, 50 µm. See also Figure S2F.

Fig 3: Nuclear CMTR1 Is Required for Dendritic Development(A) The chemical structure of capped RNAs. RNMT (mRNA cap guanine-N7 methyltransferase), CMTR1, and CMTR2, which transfer the methyl group from S-adenosylmethionine (SAM) to the corresponding positions, are indicated. SAH, S-adenosylhomocysteine.(B) Developmental expression of CMTR1 in neurons at the denoted days in vitro (DIV). The level of CMTR1 was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as a relative ratio to DIV2. Data are mean ± SEM from three cultures.(C–E) CMTR1 domain structure (C). The K239A and ?NLS mutations render catalytic dead and cytoplasm-localized CMTR1, respectively. DIV2 rat neurons were infected with lentivirus expressing siCTL or siCM1 ± RFP or RFP-tagged human CMTR1 WT or mutants. The infected neurons at DIV7 were used for immunoblotting or immunostaining of RFP and MAP2 (D and E). Scales, 50 µm.(F) Sholl analysis to count the dendritic intersections in every 10-µm segment away from the soma.(G) Total dendritic number and length are expressed as mean ± SEM (n = 60 neurons from three cultures). ***p < 0.001, two-way analysis of variance (ANOVA). See also Figures S1 and S2.

Fig 4: Knockdown of CMTR1 Decreases CaMK2a Expression to Affect Dendrite Development(A) Gene Ontology (GO) analysis of enrichment clusters from duplicate microarrays. The statistical significance of each cluster is expressed as -log10 P.(B) The list of genes from the dendrite GO cluster with most transcriptomic changes in siCM1 neurons. Ltbp1, latent transforming growth factor ß binding protein 1; Cdkn1a, cyclin-dependent kinase inhibitor 1; Pdyn, prodynorphin; Sort1, sortilin1; Atp7a, copper-transporting P-type ATPase; Camk2a, calcium/calmodulin-dependent protein kinase 2a; Ptk2b, protein tyrosine kinase 2b; Twf1, twinfilin-1; Pafah1b1, platelet-activating factor acetylhydrolase 1b subunit 1; Prkcg, protein kinase C gamma type; Kif5a, kinesin family member 5a.(C) The normalized RNA levels relative to Gapdh were determined by qRT-PCR.(D) The CaMK2a level relative to GAPDH in siCTL and siCM1 neurons ± ectopic expression of denoted mutant.(E) DIV2 neurons infected with the denoted lentivirus were immunostained with MAP2. Scales, 50 µm.(F) Sholl analysis.(G) Total dendritic number and length per neuron (n = 60). Data are mean ± SEM collected from =3 independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test in (C) and (D) and two-way ANOVA in (F) and (G). See also Figure S3.